The retinoblastoma family of proteins is a key regulator of cell cycle progression, differentiation, and apoptosis. These tumor suppressors that play a key role in oncogenesis are "active" when in a hypo-phosphorylated state. O-linked N-acetylglucosamine (O- GlcNAc) is an abundant and dynamic post-translational modification of cytoplasmic proteins. This modification that occurs on serine/threonine residues appears to be reciprocal in many ways to phosphorylation. Since preliminary data has identified O-GlcNAc on some members of the retinoblastoma family of proteins, this grant seeks to determine the functional consequences of O-GlcNAc modification of the retinoblastoma family of tumor suppressors. The following hypotheses will be tested: (1) that O-GlcNAcylation and serine/threonine phosphorylation of the retinoblastoma family of proteins occur in a reciprocal cell cycle-dependent manner; (2) that O-GlcNAc plays a critical role in protein-protein interaction; and (3) that O- GlcNAc protects the retinoblastoma family of proteins from degradation via the apoptotic caspase pathway. These hypotheses will be tested by the completion of the following specific aims: (1) to characterize the O-GlcNAc modification of Rb, RBL1 (p107), and RBL2 (p130, RB2); and (2) to elucidate the role of O-GlcNAc in retinoblastoma family of protein-protein interactions.